Pharmaceutical compositions comprising amifostine and related compounds

ABSTRACT

The present invention is directed to pharmaceutical compositions comprising amifostine or a related compound and a compound which may be selected from the group consisting of an anesthetic, a corticosteroid, an antihistamine and an anti-inflammatory. The present invention is also directed to methods of treating or protecting tissue of a human from damage associated with radiation and/or chemotherapeutic treatment of cancers by the application of the pharmaceutical compositions.

This application is a continuation of U.S. patent application Ser. No.10/872,870, filed Jun. 22, 2004, which is a divisional of U.S. patentapplication Ser. No. 09/818,555, filed Mar. 28, 2001, now U.S. Pat. No.6,753,323, issued Jun. 22, 2004, which is a continuation of U.S. patentapplication Ser. No. 09/298,824, filed Apr. 26, 1999, now U.S. Pat. No.6,239,119, issued May 29, 2001, which claims the benefit of U.S.provisional patent application No. 60/083,071, filed Apr. 27, 1998, allof which are incorporated by reference in their entirety.

1. FIELD OF THE INVENTION

The invention relates to topical use of aminothiols and compositionscontaining them for the protection from and treatment of radiationand/or chemotherapeutic damage to tissue.

2. INTRODUCTION

The present invention is directed to novel methods of using amifostineand structurally related aminothiols, including aminophosphorothioates,and their metabolites for the topical treatment of tissue. Inparticular, the invention relates to protecting mucosal, skin or hairtissue from damage by radiation and chemotherapeutic agents, as well asto the treatment of such damaged tissue. In one aspect, the inventionencompasses methods of protection of mucosal tissue, and especiallymucosal tissue of the head and neck regions, from chemical, radiation,and radio/chemo induced mucositis and conditions related to mucositis,associated with the treatment of cancers. The methods are achieved bythe topical application of amifostine, structurally related compounds ortheir metabolites. The invention also encompasses treatment andprevention of infections associated with mucositis in mucosa of the headand neck region by topical application of amifostine and relatedcompounds. Topical application of these radiochemical protectants allowsthe use of more aggressive radiochemical treatment schedules whileavoiding the toxic effects of systemically administered amifostine.

3. BACKGROUND OF THE INVENTION

3.1 Systemically Administered Amifostine

The compound S-2-(3-aminopropylamino)ethyl dihydrogen phosphorothioate(also known as amifostine, ethiofos, ETHYOL®, NSC 296961, and WR-2721,and which will hereinafter be referred to as “amifostine”) has beenknown for over thirty years, and was originally developed by the WalterReed Institute of Research as an antiradiation agent (radioprotectant).In particular, amifostine was developed for military use against x-rayor nuclear radiation which may be encountered during military conflicts.Bulk amifostine and other aminoalkyl dihydrogen phosphorothioates, andmethods to obtain them, are disclosed in U.S. Pat. No. 3,892,824, whichis incorporated herein by reference.

In addition to its utility as a military antiradiation agent, amifostinehas demonstrated excellent utility as a non-military radioprotectant andchemoprotectant when administered systemically prior to chemotherapyand/or radiation therapy. Amifostine acts to protect normal tissueagainst the adverse effects which accompany the use of radiochemicaltherapies for the treatment of various cancers, while largely leavingthe target cancerous tissues unprotected. This protective effect isobserved in radiation and chemotherapeutic treatments by, for example,alkylating agents such as cyclophosphamide, cisplatin, carboplatin,doxorubicin and its derivatives, and mitomycin and its derivatives. Forrepresentative studies, see, e.g., Constine et al., Int. J. Radia.Oncol. Biol. Phys., 12, 1505-1508 (1986); Liu et al., Cancer, 69(11),2820-2825 (1992); Wadler et al., J. Clin. Oncol., 11(8), 1511-1516(1993); and Büntzel et al., Ann. Oncol. 7 (Suppl. 5), 81, 381P (1996).

Similarly, it has been reported that amifostine may be used to protectagainst the harmful side effects of 3′-azido-3′-deoxythymidine (AZT)therapy. In addition, amifostine and its derivatives appear to exerttheir protective effects without significantly affecting the beneficialproperties of the administered therapeutic agents.

Amifostine is approved in the United States for treatment to reduce thecumulative renal toxicity associated with repeated administration ofcisplatin in patients with advanced ovarian or non-small cell lungcancer. Physicians' Desk Reference, 51 st ed. (1997).

Amifostine is a pro-drug that is dephosphorylated at the tissue site byalkaline phosphatase to the free thiol, which is the active metabolite(WR-1065). Without wishing to be bound by theory, it is believed thatonce inside the cell, the active free thiol can protect against thetoxicities associated with radiation by acting as a scavenger for oxygenfree-radicals. (See, Yuhas, in Radiation-Drug Interactions in CancerManagement, pp. 303-352 (1977); Yuhas, J. Natl. Cancer Inst., 50, 69-78(1973); Philips et al., Cancer Treat. Rep., 68, 291-302 (1984)).

Amifostine shows these favorable radioprotective effects whenadministered systemically prior to radiation treatment. Systemicadministration, however, suffers from numerous disadvantages. Thetypical systemic route of administration is intravenously, butadministering compounds intravenously is extremely inconvenient,particularly when a daily dosing schedule for several weeks isnecessary. In addition, when amifostine is administered systemically,patients suffer from dose-dependent undesirable side-effects such asnausea, vomiting, emesis and hypotension, as well as flushing or feelingof warmth, chills or feeling of coldness, dizziness, somnolence, hiccupsand sneezing. At high enough doses, systemic amifostine is toxic.

3.2 Topically Administered Amifostine

Topical administration of amifostine, if feasible, would be advantageousfor a number of reasons. The therapeutic effect of radiation isdose-dependent, so that it would be desirable in many cases to increasethe radiation dosage, or use an accelerated radiation schedule, in orderto increase the cure rate. Such increased doses of radiation, however,require corresponding increases in amifostine doses in order tocounteract the damage to normal tissue accompanying the increased oraccelerated radiation schedule. The protective effect of the compound issaid to be dependent upon the concentration of amifostine or its activemetabolite present in the normal tissue. Because of the adverse sideeffects of systemic amifostine, however, the amount that can beadministered systemically is severely limited. Topical administrationwould allow greater local control of the amifostine concentration,allowing higher local concentrations without delivery of the higherdoses to unaffected tissues and organs. To date, however, topicaladministration of amifostine has not been shown to be feasible.

The need for an effective topical radiation and chemotherapy(“radio/chemo”) protectant is especially acute in patients sufferingfrom radiation or chemically induced damage to mucosal tissue, such asmucositis and conditions associated with mucositis. As a specificexample, cancers of the head and neck are often highly localized, andwould benefit from aggressive radio/chemo treatment. The normal mucosaltissues of the head and neck region, such as the oral mucosa, aresusceptible to chemical and radiation damage. Chemical, radiation, andcombined radiation and chemical treatment act to deplete the mucosalbasal epithelium, thinning the tissue and causing inflammation,swelling, erythema and ultimately ulceration.

Ulceration of the mucosa leads to additional complications, as theexposed underlying tissue is vulnerable to infection. For example,Bourhis et al. evaluated an accelerated radiation schedule in patientssuffering from locally advanced head and neck cancers. Bourhis et al.,Int. J. Radiat. Oncol. Biol. Phys., 32(3), 747-752 (1995). In all of thepatients treated with the accelerated schedule, confluent mucositis wasobserved, and more than half of the patients required hospitalization totreat the mucositis. Similar results were reported by Delaney et al.(96% showed confluent mucositis), following a different aggressiveradiotherapy schedule. Delaney et al., Int. J. Radiat. Oncol. Biol.Phys., 32(3), 763-768 (1995). But for the sensitivity to head and neckmucosal tissue to radio/chemo damage, more aggressive therapeutictreatments including increased radiation doses and accelerated radiationschedules could be particularly effective at treating cancers in theseregions. Thus, protection of mucosal tissue of the head and neck regionswould be especially advantageous.

3.2.1 Topical Application to Non-Mucosal Tissue

Although much is known about the radioprotective effects of systemicallyadministered amifostine and related compounds, relatively little isknown about the effects of these compounds when administered topically.The few studies which have addressed topical administration haveproduced inconclusive results.

In an early study, Utley et al. found that topical administration ofamifostine in carbowax to the oral mucosa of mice subjected to wholehead irradiation prevented oral radiation death syndrome (LD50/8-10) bya factor of 1.4, with no toxicity observed at the dosages tested. Utleyet al., Int. J. Radiat. Oncol. Biol. Phys., 1, Supp. 1, No. 154 (1976).Systemically administered amifostine was found to be more effective inpreventing oral radiation death, although some deaths due to drugtoxicity were reported. The study did not address protection of othertissues or of the oral mucosa per se from mucositis.

Several studies have looked at the radioprotective effects of topicalamifostine on radiation-induced damage to non-mucosal tissue,particularly to rat and mouse skin.

In an early pre-clinical study, Lowy et al. studied the radioprotectiveeffects of systemically and topically administered amifostine in mice.Lowy et al., Radiation Biology, 105, 425-428 (1972). The amifostine wasadministered to mouse skin as a paste formed from an aqueous sodiumbicarbonate solution (pH 7) and Unibase. Although the study foundsystemically administered amifostine to be effective in reducing theseverity of radiation damage, topically applied amifostine was found toprovide no radioprotective effect at any dose studied.

Similarly, Verhey et al. found amifostine to be ineffective to protectmouse skin from radiation-induced damage when applied topically. Verheyet al., Radiation Research, 93, 175-183 (1983). A gauze saturated with a10% solution of amifostine in saline was applied to murine skin for 15to 60 minutes, followed by ¹³⁷Cs irradiation. The study found nosignificant radioprotective effects for topically-applied amifostine.

More recently, Geng et al. compared the effects of topically andsystemically administered 16, 16 dm prostaglandin E₂ (PGE₂) andamifostine on radiation-induced alopecia in mice. Geng et al., Int. J.Radiat. Biol., 61(4), 533-537 (1992); see also Malkinson et al., J.Invest. Dermatol., 101(suppl), 135S-137S (1993), reporting similarresults. In the topical studies, a 0.3 mg sample of the dephosphorylatedform of amifostine, WR-1065, in 0.2 mL Ringer's solution wasadministered to the depilated mouse skin prior to fractionatedirradiation treatment, then the rate of hair regrowth was studied as afunction of radiation dosage. Although topically administered WR-1065showed some effectiveness in protecting hair matrix cells fromradiation-induced injury, the Geng study found systemically administeredamifostine to be more effective at all radiation doses studied.

3.2.2 Topical Application to Mucosal Tissue

Several studies have examined the possibility of using amifostine orrelated compounds topically to protect intestinal mucosal tissue fromradiation damage.

Ben-Josef et al. demonstrated that amifostine applied topically by anintrarectal injection of a 2% amifostine gel results in an accumulationof the amifostine metabolite WR-1065 in the rectal wall of rats.Ben-Josef et al., Radiation Research, 143, 107-110 (1995). The study didnot, however, examine the radioprotective effect in mucosal tissue thatmight result from the accumulated WR-1065.

Montana et al. tested the effect of amifostine applied topically toprotect intestinal mucosa in patients undergoing radiation treatment ofthe pelvis. Cancer, 69(11), 2826-2830 (1992). An amifostine inProctofoam preparation was administered topically by enema at dosages of100 to 450 mg per enema 45 minutes prior to pelvic radiation treatment.The Montana et al. study found that topically applied amifostine avoidedmany of the side effects typically observed when the drug isadministered systemically. However, no significant differences werefound in rectal mucosal damage between a control group, which did notreceive amifostine, and a test group, which received the topicalamifostine treatments. Montana et al. speculated that the lack ofprotective effect may be due to the mode of administering the drug.

An opposite conclusion was reached in an animal study by Delaney et al.Delaney et al., Cancer, 74(8), 2379-2384 (1994). Delaney et al. studiedthe radioprotective effect of topical solutions of amifostine injectedinto the small intestine of rats. Amifostine was prepared in a pH 9 Trisbuffer (tris(hydroxymethyl)aminomethane) at a concentration of 150mg/mL, and administered intralumenally prior to irradiation of theexteriorized rat small bowel. The study concluded that amifostine, andparticularly amifostine in an alkaline vehicle, was an effectiveradioprotector against intestinal mucositis in rats.

Clearly, the results of these limited studies have been inconclusive andcan be considered contradictory. There is some evidence that topicalamifostine or WR-1065 may provide protection of skin or intestinalmucosa, but the studies to date show conflicting results. In addition,the studies of mucosal tissues to date have focused on intestinal mucosaand the effects of radiation induced mucosal tissue damage thataccompanies pelvic irradiation. Some studies suggest that the vehicleused to deliver the active agent may be a factor to consider indetermining potential efficacy.

Mucosal tissues of the head and neck region are particularly sensitiveto radiation and chemically-induced damage association withradiochemical treatment of head and neck cancers. Mucositis of thesetissues results in extreme patient discomfort, as well as incomplications due to infection of ulcerated mucositic tissues. There hasyet to be identified a safe and effective method of protecting themucosal tissues of the head and neck region from radiochemically-induceddamage without the undesirable side effects of systemic administrationof protectant drugs.

Thus, there is a need for a safe and effective method of protectingtissues from damage due to radio- or chemotherapeutic treatments whileavoiding the undesirable effects of systemically administeredradio/chemo protectants.

4. SUMMARY OF THE INVENTION

The present invention relates to methods of protecting or treatingtissue, skin or hair, particularly mucosal tissue, from damageassociated with radiation treatment and chemotherapy, while avoiding theundesirable side effects associated with conventional systemicadministration of radio/chemo protectant compounds.

One aspect of the invention relates to protection of tissue by topicallyadministering to the tissue prior to, during, or after, or preferablyboth prior to and during irradiation and/or chemotherapy, atherapeutically effective amount of an aminophosphorothioate oraminoalkyl thiol compound. The aminophosphorothioate or aminoalkyl thiolcompound has the formula:R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂wherein:

R₁ is hydrogen, C₅-C₇ aryl, C₂-C₇ acyl, or C₁-C₇ alkyl;

R₂ is hydrogen, PO₃H₂ or R₃, wherein R₃ is R₁NH(CH₂)_(n)NH(CH₂)_(m)S—;and

n and m are each independently an integer from 1 to 10, preferably from2 to 6.

The invention also encompasses pharmaceutically acceptable salts,solvates and hydrates of the aminophosphorothioate or aminoalkyl thiolcompounds.

In another aspect, the invention relates to protection of mucosal tissueby topically administering to the mucosal tissue prior to, during, orafter, or preferably both prior to and during irradiation and/orchemotherapy, a therapeutically effective amount of theaminophosphorothioate or aminoalkyl thiol compounds described herein.

It is surprisingly discovered that the aminophosphorothioate oraminoalkyl thiol compounds also exhibit significant antibacterialproperties. Thus, another aspect of the invention relates to a method ofprotecting tissue from bacterial infection, or treating the tissue forinfection, by topically administering to the affected tissue theaminophosphorothioate or aminoalkyl thiol compounds described herein.This is particularly advantageous given the secondary infections thatcan occur in cancer patients, particularly mucosal tissue infections inpatients undergoing treatment with radiation and/or chemotherapy.

In a further aspect, the present invention encompasses a method ofprotection or treatment of tissues from radiation or chemically induceddamage and from infection by topically administering to the tissue theaminophosphorothioate or aminoalkyl thiol compounds described herein.Such tissue includes hair, skin and mucosal tissue.

In another aspect, the invention relates to self-preserving multi-dosepharmaceutical preparations which contain an aminophosphorothioate oraminoalkyl thiol compound.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the weight loss of mice irradiated on the lip with a singledose of 16.5 Gy, in groups which received systemic amifostine, topicalamifostine, and a control group which received no amifostine

FIG. 2 shows the lip mucosal reaction scores for the irradiated mice.

6. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for methods of treatment of tissue, hairand skin, particularly mucosal tissues, to protect these tissues fromradiation and chemically-induced damage without the well-known sideeffects of systemically administered mucoprotectant compounds. Theinvention arises, in part, from the surprising discovery that theradioprotectant compound, amifostine, and structurally related compoundsand metabolites, can be effectively and safely applied topically totissues, including mucosal tissues, to provide significantradioprotection. The specific compounds which can be used, their methodsof use, and examples of their use are described in the followingsections. These include prodrugs, metabolites, derivatives and analoguesof amifostine as well as salts, solvates and hydrates of thesecompounds.

The invention also encompasses an improved therapy for the treatment ofneoplastic disorders in humans by utilizing the topical application ofthe compounds described herein before, during and/or after treatmentwith radiation or chemotherapeutics. This topical application can bothtreat and protect the patient from damage including mucositis andrelated disorders as well as bacterial infection.

6.1. Protection By Topically Applied Amifostine And Related Compounds

In one embodiment, the present invention relates to topicaladministration of one or more aminoalkyl phosphorothioate or aminoalkylthiol compounds to tissue, skin or hair, prior to, during and/or afterirradiation and/or administration of chemotherapeutic treatments.

6.1.1. Compounds

Compounds which can be advantageously administered according to themethods described herein are aminoalkyl phosphorothioate or aminoalkylthiol compounds which exhibit selective radioprotection orchemoprotection of normal tissues. Such aminoalkyl phosphorothioate oraminoalkyl thiol compounds, as well as pharmaceutically acceptable saltsand/or hydrates thereof, are either known to those of skill in the artor can be identified without undue experimentation using establishedtests routinely employed in the art.

Compounds that can be used within the present invention includeamifostine (WR-2721), as well as salts, hydrates, active metabolites,pro-drugs, and functional derivatives or analogues. More specifically,the invention includes all pro-drugs and metabolites of amifostine andpro-drugs of the active metabolites. Thus, compounds known to theskilled artisan to be suitable for administration to humans and known tobe metabolites or otherwise converted into active thiols includingmetabolites such as WR-1065 and WR-33278 (disulfide), and WR-151327 andits active thiols, including metabolites such as WR-151326 and itscorresponding disulfide, are encompassed within the present invention.

Similarly, included herein are aminothiols that exhibit activity similarto that of amifostine or its metabolites. Preferably, these compoundsare structurally related to amifostine. Alternatively, these compoundsare pro-drugs that are metabolized in vivo to a biologically activeagent. These compounds are also encompassed by the present invention.Specific examples are illustrated herein.

Aminothiol compounds which can be used in the present invention arerepresented by the following formula (I):R₁NH(CH₂)_(n)(CH₂)_(m)SR₂  (I)wherein R₁ is hydrogen, C₅-C₇ aryl, C₂-C₇ acyl, or C₁-C₇ alkyl; R₂ ishydrogen, PO₃H₂ or R₃, wherein R₃ is R₁NH(CH₂)_(n)(CH₂)_(m)S—; and n andm are each an integer from 1 to 10, preferably from 2 to 6.

The methods of the present invention also encompasses the use ofpharmaceutically acceptable salts and hydrates of the compounds offormula (I) above.

Preferred compounds useful in the methods of the invention are theS-ω(ω-amino-alkylamino)alkyl dihydrogen phosphorothioate analoguesrepresented by the formula:R—NH—(C_(n)H_(2n))—NH—(C_(m)H_(2m))—S—PO₃H₂wherein R is hydrogen or an alkyl group containing 1 to 7 carbon atomsand m and n independently have a value of from 1 to 10, preferably 2 to6.

Amifostine (WR-2721) has the structure:H₂N—(CH₂)₃—NH—(CH₂)₂—S—PO₃H₂

One preferred metabolite of amifostine is a dephosphorylated free thiolform known as WR-1065 (chemical nomenclature:S-2-(3-aminopropylamino)ethanethiol), which can be depicted as follows:H₂N—(CH₂)₃—NH(CH₂)₂—SH.

Another preferred metabolite of amifostine is its disulfide, known asWR-33278([2-[(aminopropyl)amino]ethanthiol]-N,N′-dithioidi-2,1-ethanediyl)bis-1,3-propanediamine),which can be depicted as follows:H₂N—(CH₂)₃—NH—(CH₂)₂—S—S—(CH₂)₂—NH—(CH₂)₃—NH₂.

A preferred analogue of amifostine is the compound designated asWR-151327 (chemical nomenclature:1-propanethiol-3-[[3-(methylamino)propyl]amino]-dihydrogenphosphothiorate), which can be depicted as follows:CH₃NH(CH₂)₃NH(CH₂)₃SPO₃H₂.

Another preferred analogue of amifostine is the compound designatedWR-151326, a dephosphorylated free thiol form of WR-151327 having thechemical structure:CH₃NH(CH₂)₃NH(CH₂)₃SH.

Other specific compounds suitable for use in the present inventioninclude, but are not limited to:

-   S-1-(aminoethyl)phosphorothioic acid (WR-638);-   S-[2-(3-methylaminopropyl)aminoethyl]phosphorothioate acid    (WR-3689);-   S-2-(4-aminobutylamino)ethyl phosphorothioic acid (WR-2822);-   3-[(2-mercaptoethyl)amino]propionamide p-toluene-sulfonate    (WR-2529);-   S-1-(2-hydroxy-3-amino)propyl phosphorothioic acid (WR-77913);-   2-[3-(methylamino)propylamino]ethanethiol (WR-255591);-   S-2-(5-aminopentylamino)ethyl phosphorothioic acid (WR-2823);-   1-[3-(3-aminopropyl)thiazolidin-2-Y1]-D-gluco-1,2,3,4,5pentane-pentol    dihydrochloride (WR-255709).

Additional aminothiols suitable for use in the present inventioninclude, but are not limited to, S-2-(3-ethylaminopropylamino)ethyldihydrogen phosphorothioate, S-2-(3-aminopropylamino)-2-methylpropyldihydrogen phosphorothioate, S-2-(2-aminoethylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(4-aminobutylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(5-aminopentylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(6-aminohexylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(2-methylaminoethylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(3-methylaminopropylamino)-2-ethyl dihydrogenphosphorothioate, and S-3-(3-methylamino-propylamino)-3-propyldihydrogen phosphorothioate (WR-151327) and pharmaceutically acceptablesalts thereof. Preferably, the aminothiol is amifostine, WR-1065,WR-33278, WR-151327 or WR-151326; most preferably it is amifostine.

Amifostine, and many of its salts, analogues and derivatives thereofsuitable for use in the methods of the invention are commerciallyavailable, or can easily be prepared using standard techniques. Theaminothiol compounds useful in the methods of the invention may beprepared by methods known in the art (see, e.g., Cortese, 1943, OrganicSynthesis pp. 91-93, Coll. Vol. II, Blatt, Ed., John Wiley & Sons, Inc.,New York, N.Y.; Akerfeldt, 1960, Acta Chem. Scand. 14:1980; Piper etal., 1966, Chem. Ind. (London): 2010). Certain aminothiol compounds, aswell as methods of synthesizing such compounds, are described in detailin U.S. Pat. Nos. 3,892,824, 5,424,472 and 5,591,731, and WO 96/25045.

The aminothiol compounds useful in the methods of the invention may bein the form of free acids, free bases, or pharmaceutically acceptableaddition salts thereof. Such salts can be prepared by treating anaminothiol compound with an appropriate acid and/or base. Such acidsinclude, by way of example and not limitation, inorganic acids such ashydrohalic acids (hydrochloric, hydrobromic, hydrofluoric, etc.),sulfuric acid, nitric acid, phosphoric acid, etc. and organic acids suchas acetic acid, propanoic acid, 2-hydroxyacetic acid, 2-hydroxypropanoicacid, 2-oxopropanoic acid, propandioic acid, butandioic acid, etc.Conversely, the salt can be converted into the free base form bytreatment with alkali.

The aminothiol compounds useful in the methods of the invention, as wellas the pharmaceutically acceptable addition salts thereof, may be in ahydrated, solvated or anhydrous form. Methods of preparing such formswill be apparent to those of skill in the art of organic chemistry.

In another preferred embodiment, the compound is a salt of WR-1065,preferably a succinate, a pamoate, a bis(trifluoroacetate) or adi-hydrochloric acid salt. The solubilities of these salts are shown inthe Examples section below.

6.1.2 Tissues to be Treated or Protected

It should be recognized that any and all tissue, skin or hair can betreated or protected topically in accordance with the present invention.However, the preferred tissues to be treated or protected according tothe methods of the present invention are mucosal tissues. These tissuesinclude, but are not limited to, alveolar, esophageal, gastric,gingival, laryngeal, lingual, nasal, olfactory, oral, pharyngeal,respiratory, tracheal and vaginal mucosa, as well as mucosa of theauditory tube, bronchi, ductus deferens, urethra, gallbladder, seminalvesicle, small intestine, tympanic cavity, ureter, urinary bladder anduterine tube. More preferred are mucosal tissues of the head and neckregion, which include but are not limited to, esophageal, gingival,laryngeal, lingual, nasal, olfactory, oral, pharyngeal and trachealmucosa, as well as mucosa of the tympanic cavity.

The invention is particularly well-suited to prevent or treat damage tothe mucosal tissues of the oral cavity to prevent or treat mucositis andrelated conditions and complications, including severe dry-mouth knownas xerostomia. Thus, oral mucosal tissues are most preferred.

The term “protect” as used herein means to avoid, reduce the incidenceof, or reduce the severity of mucositis and related conditions andcomplications and their symptoms.

The term “treat” as used herein means to lessen or reverse the symptomsof mucositis and related conditions and complications.

6.1.3 Compositions and Formulations

The aminophosphorothioate or aminoalkyl thiol compounds describedherein, or pharmaceutically acceptable salts and/or hydrates thereof,may be administered singly, in combination with other compounds of theinvention, and/or in combination with other therapeutic agents,including cancer chemotherapeutic agents. The active compound(s) may beadministered alone or in the form of a pharmaceutical composition,wherein the active compound(s) is in admixture with one or morepharmaceutically acceptable carriers, excipients or diluents.

Suitable excipients, carriers and diluents are well known to thoseskilled in the pharmaceutical arts, and include those which are suitablefor formulating topical preparations. When the tissues to be treated aremucosal tissues, including oral mucosa, suitable excipients, carriersand diluents must also be safe for application to oral and relatedmucosal tissue. These are well known to the skilled artisan.

Examples of excipients, carriers and diluents include, for example,water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof to form lotions, tinctures, creams, emulsions, gelsor ointments, which are non-toxic and pharmaceutically acceptable. Theskilled artisan can readily select the particular excipients, carriersand diluents appropriate to the type and location of the tissue to betreated.

Additionally, moisturizers or humectants can be added to the presentcomposition if desired. Examples of such additional ingredients can befound in Remington's Pharmaceutical Sciences, 16th and 18th eds., MackPublishing, Easton Pa. (1980 & 1990).

In addition to these and other vehicles which are known to those ofordinary skill in the art, it will be understood that the pharmaceuticalcompositions of the present invention may optionally include otheringredients such as analgesics, anesthetics, antibacterial, antiyeastagents, antifungal agents, antiviral agents, antidermatitis agents,antipruritic agents, other anti-inflammatory agents,antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents,antisebborrheic agents, antihistamine agents, vitamins, corticosteroids,hormones, retinoids, topical cardiovascular agents, clotrimazole,ketoconazole, miconozole, griseofulvin, hydroxyzine, diphenhydramine,pramoxine, lidocaine, procaine, mepivacaine, monobenzone, erythromycin,tetracycline, clindamycin, meclocyline, hydroquinone, minocycline,naproxen, ibuprofen, theophylline, cromolyn, albuterol, retinoic acid,13-cis retinoic acid, hydrocortisone, hydrocortisone 21-acetate,hydrocortisone 17-valerate, hydrocortisone 17-butyrate, betamethasonevalerate, betamethasone diproprionate, triamcinolone acetonide,fluocinonide, clobetasol, proprionate, benzoyl peroxide, crotamiton,propranolol, promethazine, vitamin A palmitate, vitamin E acetate andmixtures thereof.

Depending upon the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith the active component(s). For example, penetration enhancers may beused to assist in delivering the active component, e.g., amifostine, tothe tissue. Suitable penetration enhancers include acetone, variousalcohols (e.g. ethanol, oleyl, tetrahydrofuryl, etc.), alkyl sulfoxidessuch as dimethyl sulfoxide, dimethyl acetamide, dimethyl formamide,polyethylene glycol, pyrrolidones such as polyvinylpyrrolidone, Kollidongrades (Povidone, Polyvidone), urea, and various water-soluble orinsoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60(sorbitan monostearate).

The pH of the composition, or of the tissue to which the compound(s) isapplied, may also be adjusted to improve delivery of the compound.Preferably, the pH is somewhat basic, as a basic pH is believed toenhance the stability of the active compounds. A pH of about 8 to 9 ispreferred. Similarly, the polarity of the solvent, its ionic strength ortonicity may be adjusted to improve delivery. In addition, compoundssuch as stearates may be added to compositions comprising the activecompound(s) to advantageously alter the hydrophilicity or lipophilicityof the compound(s) and improve skin delivery. In this regard, stearatescan serve as a lipid vehicle for the formulation, as an emulsifyingagent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Also, different salts, hydrates or solvatesof the active compounds may be used to further adjust the properties ofthe resulting composition.

The compounds can be formulated in compositions such as creams, lotions,ointments, gels, solutions, suspensions or other forms known to one ofskill in the art and described in, for example, Remington'sPharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa.(1980 & 1990), and Introduction to Pharmaceutical Dosage Forms, 4th ed.,Lea & Febiger, Philadelphia (1985). Most compositions of the presentinvention may be formulated as a solution, gel, lotion, cream orointment in a pharmaceutically acceptable form. Actual methods forpreparing pharmaceutical compositions are known or apparent to thoseskilled in the art and are described in detail in, for example,Remington's Pharmaceutical Sciences, 16th and 18th eds., MackPublishing, Easton Pa. (1980 & 1990).

In a preferred embodiment for use in treating mucosal tissues within theoral cavity, the active ingredient is formulated into a solutionsuitable for use as a mouthwash or as an oral gel.

Several preferred example formulations suitable for application tomucosal tissues are given in the Examples below.

6.1.4 Methods and Dosages

The compounds or pharmaceutical compositions containing them are appliedtopically to the mucosal tissue. The term “topical” as used hereinincludes any route of administration that enables the compounds to linethe mucosal tissue; i.e., it includes any route of administration otherthan parenteral or per os. Topical includes, for example, sprayapplication, smearing, soaking and gargling.

The specific form of the compounds and their manner of administrationdepends in part upon the particular tissue to be treated. The compoundsor pharmaceutical compositions containing them can be applied, forexample, as a mouthwash to coat the oral mucosal tissue, as a spray orsyringe to coat the mucosal tissues of the nose and/or throat, or as acream or paste, an enema, or other forms of topical administration knownto one of skill in the art, as appropriate.

The amount of compound to be delivered, as well as the dosing schedulenecessary to provide the desired radio/chemo protective effects, will bedependent upon the bioavailability of the specific compound selected(and/or an active metabolite thereof), the disorder being treated, theradiation and/or chemotherapeutic dosage schedule, and other factorsthat will be apparent to those of skill in the art.

When the compound is amifostine, preferably it will be administered at adosage of about 10 to 600 mg per dose, more preferably at 100 to 300 mgper dose, and most preferably about 200 mg per dose. It should beunderstood that the preferred dosages are for localized administration,and if protection for a large amount of tissue is desired, the dosageshould be adjusted accordingly. The most preferred dosage of 200 mg perdose is appropriate, for example, for a typical application such as amouthwash to protect oral mucosal tissues.

For topical application, it is convenient to provide the active compoundin a pharmaceutical preparation at a concentration which will facilitateeasy application of the appropriate dosage, i.e., the concentrationshould be chosen so that the volume of preparation to be applied is nottoo great or too small. For typical treatment, a convenient volume fortopical application is about 1 to 30 mL per application. Thus, forexample, when the desired dosage is 200 mg, it is convenient toformulate the compound in a preparation at a concentration of about 20mg/mL, so that the desired dosage is administered by topically applyingabout 10 mL of the preparation. However, in particular situations it maybe desirable to formulate the compound in a smaller or larger volume.One of skill in the art can readily determine a convenient concentrationfor a desired dosage.

While the dosage ranges given above are the preferred ranges foramifostine, these ranges should be adjusted according to the molecularweight of the active compound to deliver an equivalent number of moles.Thus, for example, a dosage of about 200 mg amifostine is equivalent toa dosage of about 130 mg WR-1065 if all other factors are equivalent.

The compounds may be administered prior to, during, or subsequent toradiation or chemotherapeutic treatment. Preferably, in order to protectagainst tissue damage, the compounds will be administered to the tissueup to about 90 minutes prior to each radiation or chemotherapytreatment, and more preferably up to about 30 minutes prior to eachradiation or chemotherapy treatment.

The administration can be intermittent or continuous, and can continueor resume during or after treatment. Thus, for example, with specificamounts and compounds given only by way of illustration, for thetreatment of oral mucosa about 10 mL of a mouthwash formulationcontaining about 20 mg/mL of amifostine may be administered to a patient5 to 30 minutes before irradiation or before administration of achemotherapeutic agent. The formulation can be rinsed and/or gargled inthe mouth for about 10 seconds to about 2 minutes, then spit out.Additional dosages administered in a similar manner can be given duringtreatment as, for example, between different chemotherapeutic agents ina regimen incorporating multiple chemotherapeutic agents per treatment.

Alternatively, the compound may be administered in a gel, lotion,ointment or other suitable form which is applied to the tissue up toabout 90 minutes before irradiation or chemotherapeutic treatment andremains on the tissue during the treatment.

The same dosage and concentrations can also be used when the compound isadministered after irradiation and/or chemotherapeutic treatment. Itshould be understood that each of the three administrations (before,during and after radio- and/or chemotherapy treatment) may be usedalone, or in any combination of two or all three administrations, asneeded.

6.2. Antibacterial Properties of Amifostine And Related Compounds

The present invention also encompasses methods of preventing andtreating infections, particularly those associated with mucositis, suchas secondary infections that occur as a result of radiation and/orchemotherapy. Bacterial infection of mucosal tissues is a common sideeffect of radiation and chemical damage associated with radio/chemocancer treatments. Damage to the mucosal basal epithelium, and resultantulceration, leaves the exposed underlying tissues extremely susceptibleto bacterial infection.

It is surprisingly discovered that amifostine and related compounds arebroad spectrum antibiotics as described in the United StatesPharmacopeia, 23rd ed. (1995), “AntimicrobialPreservatives—Effectiveness” test protocol. Thus, pharmaceuticalpreparations containing amifostine and related compounds can beself-preserving. This self-preserving property is particularlyadvantageous for use in multi-dose formulations.

The antibacterial properties can also be used advantageously to preventand treat infections, particularly those associated with mucositis, whenthe amifostine compounds are applied topically. The antibacterialproperties allow the topical use of the amifostine compound afterirradiation or chemotherapy to protect against bacterial infection aswell as symptoms of mucositis.

The types of secondary bacterial infections that can be readily treatedinclude gram negative, gram positive, yeast, mold and pseudomonasinfections.

Certain embodiments of the invention are illustrated, and not limited,by the following working examples.

7. EXAMPLES 7.1. Example 1 Effect of Topical Administration ofAmifostine on Radiation-Induced Mucositis in Mice

The effect of topical amifostine on murine oral mucosa exposed toradiation was studied, to determine the radioprotective effects oftopically and systemically applied amifostine.

Materials and Methods

The experimental model used was the inferior lip mucosa of the mice. Theexperimental murine model was developed by Parkins et al., Radiother.Oncol., 1, 159-165 (1983), which has been shown to be a reproducibletool to study mucosal reactions after irradiation in mice. C57BL/6 mice8-10 weeks old were used and fed with semi-liquid food. Unanesthetizedmice were maintained in supine position and irradiated exclusively onthe tip of the mouth. The mice were immobilized in jigs comparable tothose described in Ang et al., Int. J. Radiat. Oncol. Biol. Phys., 8,145-148 (1982). The irradiation was performed with an RT 250 Phillipsapparatus, delivering 1.98 Gy/min (200 kV, 20 mA, filter of 0.2 mm Cu).During irradiation, a constant normobaric air renewal was maintained.

For the systemic applications, amifostine was dissolved in a 9% NaClsolution 5 minutes before systemic IP injection of 200 to 400 mg/kg,which was administered 30 minutes before irradiation. A placebo solutionof 9% NaCl was used for a control group. For the topical applications,50 mg of amifostine was dissolved in 1 mL of a solution containing 1%stearate, 5% H₂O, and ammonia to adjust the pH of the solution to 8.8.The topical application was administered to the lip mucosa of the micefor a period of 30 minutes before each radiation session, and wasmaintained throughout the duration of irradiation. A single dose of 16.5Gy, and a fractionated schedule delivering 24 Gy in 4 fractions over 2days (with 8 hour intervals between fractions) were administered.

The effect of irradiation on lip mucosa was evaluated using the scoringsystem described by Parker, and shown in Table 1. TABLE 1 Parkinsscoring system Score Mucosal Observation/Erythema   0.5 doubtful ifabnormally pink 1 slight but definite reddening 2 severe reddening 3focal desquamation 4 exudation or crusting covering about ½ lip area 5exudation or crusting covering more than ½ lip area Separate score foredema (swelling) of the lips, to be added to the erythema scores givinga maximum score of 7: Score Mucosal Observation/Edema   0.5 50-50doubtful if any swelling 1 slight but definite swelling 2 severeswelling

Four groups of eight mice were scored and weighed each day. Any micehaving lost 30% or more of the initial weight was sacrificed. The effectof mucositis and weight loss were compared at the maximum of the acutereactions (day 11) among the different groups receiving or not receivingamifostine treatments, using the Mann and Whitney test.

Results

In a first set of experiments, the mice were irradiated with a singledose of 16.5 Gy. The distribution of weight loss and of the score ofmucosal reactions after IP and topical administration is shown inFIG. 1. The maximum mucosal reactions were observed on day 11, followingthe start of irradiation. The mucosal reaction grades of the mice areshown in FIG. 2. The grades of mucosal reactions were significantlylower in the groups receiving amifostine compared to the control group.The Figure shows that the mucosal grades for IP administered amifostineare lower, but the difference was not found to be statisticallysignificant.

Similar results were found in a second experiment, in which afractionated schedule of 24 Gy in 4 fractions over 2 days was deliveredusing two different levels of amifostine IP: 200 mg/kg and 400 mg/kg.400 mg/kg given before each fraction of radiation was the maximumtolerated dose. In this experiment, the topical amifostine wasadministered as described above without modification.

The maximum grade of mucosal reactions occurred on day 11 afterirradiation, and was found to be 3.5±0.2 and 2.5±0.4 for the 200 and 400mg/kg IP dosages, respectively. The error bars represent the standarderror. For topical amifostine, the maximum grade of mucositis was foundto be 3.9±0.2, and was not statistically significantly different fromthe IP groups. The control group receiving no amifostine showed amucosal reaction grade of 5.6±0.4, and was statistically different(p=0.001) from the amifostine groups.

7.2. Example 2 Antibacterial Properties of Amifostine

Amifostine was studied for its ability to inhibit microbial growth.Clear 10 cc vials, stoppers and seals were sterilized for use in thestudy. Two separate procedures were performed: a refrigerated storagestudy (2° to 8° C.) and a room temperature storage study (20° to 25°C.). For each test portion, positive controls were prepared for eachorganism by placing 9.5 mL sterile 0.9% sodium chloride into separatesterile vials and inoculating each with 10³ to 10⁴ of one of the testorganisms. In addition, amifostine test specimens were prepared for eachtest portion by reconstituting six ETHYOL® vials with 0.9% sodiumchloride. the contents of each of these vials were then transferred toclear sterile vials to allow for easier visual examination. One vial wasdesignated as a negative control, and each of the remaining five vialswas inoculated with 10³ to 10⁴ of one of the test organisms. All vialswere protected from light throughout the test to simulate the amberETHYOL® vial.

Test Performance

Refrigerated Storage (2°-8° C.)

Positive controls, product test specimens, and a negative control wereprepared. At this time, population counts were performed on eachpositive control. Population counts were also performed on positivecontrols and product test specimens at 24, 48 and 72 hours. Thesesamples were diluted with sterile water and plated in duplicate with atarget of 30 to 300 colonies per plate. Candida albicans, Escherichiacoli, Pseudomonas aeruginosa, and Staphylococcus aureus were overpouredwith TSA agar and incubated at 30-35° C. for 48 hours. Aspergillus nigerwas overpoured with Sabouraud agar and incubated at 20-25° C. for 48hours. The plates were counted and the population counts are summarizedin Table 2. TABLE 2 Refrigerated Storage (2°-8° C.) Time CFU/mL in %CFU/mL in (hr) Amifostine Change Saline % Change S. aureus 0 9.50 × 10¹*9.50 × 10¹ 24 4.75 × 10¹ −50.0% 4.75 × 10¹ −50% 48 4.75 × 10¹ −50.0%4.75 × 10¹ −50% 72 4.75 × 10¹ −50.0% 4.75 × 10¹ −50% P. aeruginosa 03.80 × 10³* 3.80 × 10³ 24 3.04 × 10³ −20.0 3.47 × 10³ −0.08 48 1.76 ×10³ −53.7 1.48 × 10³ −61.0 72 1.85 × 10³ −51.3 2.23 × 10³ −41.3 E. coli0 9.50 × 10²* 9.50 × 10² 24 2.85 × 10² −70.0 1.90 × 10² −80.0 48 0 −1004.75 × 10¹ −95.0 72 0 −100 0 −100 C. albicans 0 1.08 × 10⁴* 1.08 × 10⁴24 3.99 × 10³ −63.0 7.70 × 10³ −28.7 48 2.09 × 10³ −80.6 5.52 × 10³−48.9 72 7.13 × 10³ −34.0 5.70 × 10³ −47.2 A. niger 0 3.00 × 10⁵* 3.00 ×10⁵ 24 1.78 × 10⁵ −40.7 1.57 × 10⁵ −47.7 48 1.67 × 10⁵ −44.3 1.38 × 10⁵−54.0 72 1.67 × 10⁵ −44.3 1.53 × 10⁵ −49.0*theoretical

At 48 hours, the concentrations of viable microorganisms were reduced by≧44.3% of the initial concentration. Incubation for an additional 24hours demonstrated continued loss of viability.

Room Temperature Storage (20°-25° C.)

Positive controls, product test specimens, and a negative control wereprepared. At this time, population counts were performed on eachpositive control. Population counts were also performed on positivecontrols and product test specimens at 24 and 48 hours. These sampleswere diluted with sterile water and plated in duplicate with a target of30 to 300 colonies per plate. Candida albicans, Escherichia coli,Pseudomonas aeruginosa, and Staphyloccus aureus were overpoured with TSAagar and incubated at 30-35° C. for 48 hours. Aspergillus niger wasoverpoured with Sabouraud agar and incubated at 20-25° C. for 48 hours.The plates were counted and the population counts are summarized inTable 3. TABLE 3 Room Temperature Storage (20°-25° C.) Time CFU/mL in %CFU/mL in (hr) Amifostine Change Saline % Change S. aureus 0 1.00 × 10⁵*1.00 × 10⁵ 24 9.60 × 10⁴ −4.0% 1.01 × 10⁵   0% 48 3.32 × 10² −99.7% 0−100% P. aeruginosa 0 2.61 × 10³* 2.61 × 10³ 24 5.22 × 10² −80.0 2.09 ×10³ −21.0 48 0 −100 1.33 × 10³ −49.0 E. coli 0 2.79 × 10⁴* 2.79 × 10⁴ 242.28 × 10³ −91.8 2.90 × 10³ −89.6 48 8.55 × 10² −96.9 9.50 × 10¹ −99.6C. albicans 0 1.66 × 10⁴* 1.66 × 10⁴ 24 1.43 × 10⁴ −13.8 1.66 × 10⁴ 0 481.00 × 10⁴ −39.8 1.44 × 10⁴ −13.2 A. niger 0 9.10 × 10⁶* 9.10 × 10⁶ 241.56 × 10⁷ +58.3 1.73 × 10⁷ +52.6 48 3.52 × 10⁶ −61.3 5.46 × 10⁶ −40.0*theoretical

At 48 hours, a population count was performed on the negative controland all test samples were subcultured for microbial identification.Results revealed that the negative control was absent of microbialgrowth and all test specimens contained the stated test organisms.

At 24 hours, the concentration of viable bacteria were reduced in fourof the five compendial organisms. The concentration of A. nigerincreased at 24 hours, but decreased significantly at 48 hours.

7.3. Example 3 Antibacterial Properties of WR-1065

WR-1065 was evaluated according to a protocol based on the“Antimicrobial Preservatives-Effectiveness” Test, outlined in the UnitedStates Pharmacopeia, 23rd ed. (1995).

Procedure

Preparation of Mold Spore Inoculum Suspension

A sterile swab is used to spread 1.0 mL of a spore suspension over thesurface of solidified SDA in a 150×15 mm Petri dish so as to result in aconfluent lawn of mold growth. The culture is then incubated at 20-25°C. for 7 days to allow for mature spore development. Followingincubation, the spores are harvested in approximately 25 mL of sterile0.9% Saline with POLYSORBATE® 80 by spreading the fluid across thesurface of the culture and gently scraping with a sterile spoonula todislodge the spores. The resulting suspension is transferred into asterile tissue grinder and the piston is reciprocated several times tobreak up spore chains. A spore count is obtained with the aid of ahemocytometer and the spore concentration is adjusted, if necessary, toapproximately 1.0×10⁸ spores/mL.

Preparation of the Bacterial and Yeast Inoculum Suspensions

Bacterial challenge organisms are inoculated onto TSA slants from stockcultures and incubated for 18-24 hours at 30-35° C. The yeast challengeorganism is inoculated onto a SDA slant from stock culture, andincubated for 48 hours at 20-25° C. Following incubation, organisms areharvested from the agar surfaces in 0.9% Saline. Cell population in eachsuspension is estimated by determining percent transmittance on aspectrophotometer and consulting established growth curves for eachorganism. Each suspension is adjusted to an approximate concentration of1.0×10⁸ Colony Forming Units (CFU) per mL. The number of viablemicroorganisms in each inoculum suspension is determined by plate countprocedure, and the initial concentration of microorganisms per gram (ormL) of WR-1065 is calculated. The numbers obtained from this procedureprovide the theoretical concentration of challenge organisms present ineach test article at the start of the study, and are used as thebaseline for calculating the reduction of the number of organisms overtime.

Neutralization

The neutralizer medium used as a diluent is AOAC Neutralizer Blanks. MCTagar is used as the plating medium for all bacteria recovery. SDA/L isused as the plating medium for fungal recovery.

A neutralization study is conducted to evaluate the preservativeneutralizing ability of the media used to conduct the test and to assurethat viable organisms can be detected under the conditions of the test.Separate 1:10 (and 1:100, if necessary) dilutions of WR-1065 areprepared in diluent for each test organism. A blank control of the samevolume is also prepared for each organism. Each dilution and controltube is inoculated with 0.1 mL of organism suspension to result in alevel of approximately 10-100 CFU/mL of suspension. Plate counts areobtained on each suspension. After incubation, colony forming units areenumerated and percent recovery between control plates and test platesis evaluated. Neutralization is effective if 50% or greater recovery isobserved on test plates as compared to the inoculum control plates.

Plating and Plate Incubation

Plates prepared in this study are incubated according to the followingstandard:

-   -   MCT plates for bacteria recovery: 2-3 days at 30-35° C.    -   SDA/L plates for fungal recovery: 5-7 days at 20-25° C.

All plating for this study is conducted in duplicate.

Initial Count Procedure

Prior to inoculation of the test article, a 1:10 dilution is prepared indiluent and 1.0 mL is plated. Additional serial dilutions are preparedand plated as necessary. Two separate sets are prepared as above. Oneset is prepared for bacteria counts, and one set is prepared foryeast/mold counts.

Inoculation of Test Article

Five separate 20 g (or 20 mL, as appropriate) portions of the testarticle are placed into sterile 50 mL polypropylene conical tubes andinoculated with 0.1 mL of individual inoculum suspension to result in afinal concentration of between 1.0×10⁵ and 1.0×10⁶ CFU/g (or mL) of testarticle. All inoculated specimens are vigorously mixed to assure evendispersal of the inoculum. Inoculated test articles are held at 20-25°C. for the duration of the study.

Post Inoculation Procedure

Enumeration of viable bacterial and fungal populations in the inoculatedtest article is conducted at 7, 14, 21 and 28 days post inoculation. Ateach time interval, a 1.0 g (or mL) specimen is removed from eachinoculated specimen and plate counts are obtained by preparing serialdilutions in 9 mL tubes of diluent and plating aliquots from eachdilution. Following incubation, plates are observed and the CFU's areenumerated. The number obtained is multiplied by the dilution factor togive the number of viable organisms per g (or mL) of test article ateach time interval. At each time interval, the inoculated product isexamined and any changes observed in the appearance of the test articleduring the challenge period are recorded.

WR-1065 was tested according to the procedure described above. Theresults are shown in Table 4. TABLE 4 Test Organism Recovery (CFU/g)Test Inoculum Organism Counts* 7 Day 14 Day 21 Day 28 Day E. coli 4.9 ×10⁵ <10 <10 <10 <10 ATCC 8739 P. 5.5 × 10⁵ <10 <10 <10 <10 aeruginosaATCC 9027 S. aureus 4.1 × 10⁵ 8.8 × 10⁴ <10 <10 <10 ATCC 6538 C.albicans 4.5 × 10⁵ 3.0 × 10⁴ <10 <10 <10 ATCC 10231 A. niger 3.5 × 10⁵1.7 × 10⁵ 1.3 × 10⁵ 1.2 × 10⁵ 7.5 × 10⁴ ATCC 16404*Calculated CFU/g of sample

The initial counts of bacteria, yeast and mold were <10 CFU/g.

Interpretation

The efficacy of WR-1065 was evaluated according to the followingcriteria stated in the United States Pharmacopeia, USP 23, 1995:

The preservative is effective in the product examined if: (a) theconcentrations of viable bacteria are reduced to not more than 0.1% ofthe initial concentration by the 14th day; (b) the concentrations ofviable yeast and mold remain at or below the initial concentrationsduring the first 14 days; and (c) the concentration of each testorganism remains at or below these designated levels during theremainder of the 28 day test period.

According to these criteria, WR-1065 is an effective preservative foreach of the tested microorganisms.

7.4. Example 4 Solubility of WR-1065 Salts

As mentioned above, salts of amifostine and related compounds areincluded within the scope of the invention. Certain salts may haveadvantages in topical administration. Several salts of WR-1065 wereprepared, and their solubility in various solvents was measured. Thedata are shown in Table 5. TABLE 5 Solubility of WR-1065 Salts Est.Solubility at Est. Solubility HPLC Salt M.W. (g/mol) Solvent Saturation(mg/mL) Assay (mg/mL) Succinate 252 Water 854 715 Succinate (as free 455381 base) Succinate Ethanol insoluble 1.95 Succinate (as free 1.04 base)Pamoate 522.6 70% isopropyl 21 18.5 alcohol Pamoate (as free base) 5.344.8 Pamoate Ethanol insoluble 0.2 Pamoate (as free base) 0.05 Bis TFA*362.3 Water 820 954 Bis TFA* (as free 354 353 base) Bis TFA* Ethanol 313327 Bis TFA (as free 116 121 base) di-HCl Water 849 di-HCl (as freebase) 550*Trifluoroacetate

7.5 Example 5 Formulations

Several pharmaceutical ointments were prepared for use with amifostinefor topical application to oral mucosal tissue. Each of theseformulations has a pH of 8.8, adjusted by addition of ammonia. Theointment bases are summarized in Table 6. TABLE 6 Ointment BaseCompositions Base # Component Amount 1 Tween 80 5 mL Glycerol starch*100 g Water (sterile, demineralized) 5 mL Ammonia to pH 8.8 2 Tween 80 5mL Glycerol starch 100 g Stearates 1 g Ammonia to pH 8.8 3 Tween 80 5 mLGlycerol starch* 100 g Stearates 1.5 g Ammonia to pH 8.8 4 Lactose 10 gWater (sterile, demineralized) 10 mL Petrolatum 60 g Ammonia to pH 8.8

Tween 80 is a water-soluble ester (polyoxyethylene oxide sorbitanmonooleate, or polysorbate 80). Glycerol starch is a gelatinous mixtureof glycerol and starch, suitable as a base for a gel or ointment. Othersimilar components may be used as well.

Compositions according to the present invention were prepared by firstmixing amifostine in a minimal quantity (40 μL for 50 mg of amifostine)of a 9% sodium chloride solution, then combining with the ointment base.Formulations were prepared at amifostine concentrations of 20, 100 and200 mg/mL of ointment base.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

All references cited in the present application are incorporated byreference in their entirety.

1. A pharmaceutical composition comprising: (a) a therapeuticallyeffective amount of at least one compound of formula (I):R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂  (I) wherein R₁ is hydrogen, C₅-C₇ aryl,C₂-C₇ acyl, or C₁-C₇ alkyl; R₂ is hydrogen, PO₃H₂ or R₃, wherein R₃ isR₁NH(CH₂)_(n)NH(CH₂)_(m)S—; and n and m are each independently aninteger from 1 to 10, or a pharmaceutically acceptable salt, solvate orhydrate thereof; and (b) a second compound selected from the groupconsisting of an anesthetic, a corticosteroid, an antihistamine and ananti-inflammatory.
 2. The pharmaceutical composition of claim 1, whereinthe second compound comprises an anesthetic.
 3. The pharmaceuticalcomposition of claim 2, wherein the anesthetic comprises pramoxine,lidocaine, procaine, mepivacaine or a combination thereof.
 4. Thepharmaceutical composition of claim 1, wherein the second compoundcomprises a corticosteroid.
 5. The pharmaceutical composition of claim4, wherein the corticosteroid comprises hydrocortisone, hydrocortisone21-acetate, hydrocortisone 17-valerate, hydrocortisone 17-butyrate,betamethasone valerate, betamethasone diproprionate, triamcinoloneacetonide, fluocinonide, clobetasol proprionate or a combinationthereof.
 6. The pharmaceutical composition of claim 1, wherein thesecond compound comprises an antihistamine agent.
 7. The pharmaceuticalcomposition of claim 6, wherein the antihistamine agent compriseshydroxyzine, diphenhydramine, propranolol, promethazine, vitamin Apalmitate or a combination thereof.
 8. The pharmaceutical composition ofclaim 1, wherein the second compound comprises an anti-inflammatoryagent.
 9. The pharmaceutical composition of claim 8, wherein theanti-inflammatory agent comprises naproxen, ibuprofen, theophylline,cromolyn, albuterol, retinoic acid, 13-cis retinoic acid or acombination thereof.
 10. The pharmaceutical composition of claim 1further comprising a pharmaceutically acceptable excipient, carrier ordiluent.
 11. The pharmaceutical composition of claim 1, wherein R₁ ishydrogen or methyl; R₂ is hydrogen or PO₃H₂; m is 2 or 3; and n is 3.12. The pharmaceutical composition of claim 1, wherein the compound isamifostine, WR-1065, WR-151326, WR-151327, or WR-33278, or apharmaceutically acceptable salt, solvate or hydrate thereof.
 13. Thepharmaceutical composition of claim 12, wherein the compound of formula(I) is amifostine, or a pharmaceutically acceptable salt, solvate orhydrate thereof.
 14. The pharmaceutical composition of claim 1, whereinthe pharmaceutical composition is a cream, lotion, ointment, gel,solution, or suspension.
 15. The pharmaceutical composition of claim 14,wherein the pharmaceutical composition is a solution.
 16. Thepharmaceutical composition of claim 15, wherein the solution furthercomprises a diluent.
 17. The pharmaceutical composition of claim 16,wherein the diluent is water, acetone, ethanol, ethylene glycol,propylene glycol, butane-1,3-diol, isopropyl myristate, isopropylpalmitate, mineral oil or a combination thereof.
 18. The pharmaceuticalcomposition of claim 17, wherein the diluent is water.
 19. Thepharmaceutical composition of claim 18, wherein the water diluentfurther comprises sodium chloride, potassium chloride, calcium chlorideor a combination thereof.
 20. The pharmaceutical composition of claim 1,wherein the therapeutically effective amount of the compound of formula(I) is 10-600 mg.
 21. A method of protecting tissue of a human fromdamage caused by radiation and/or chemotherapeutic treatment, whichcomprises administering: (a) a therapeutically effective amount of atleast one compound of formula (I):R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂  (I) wherein R₁ is hydrogen, C₅-C₇ aryl,C₂-C₇ acyl, or C₁-C₇ alkyl; R₂ is hydrogen, PO₃H₂ or R₃, wherein R₃ isR₁NH(CH₂)_(n)NH(CH₂)_(m)S—; and n and m are each independently aninteger from 1 to 10, or a pharmaceutically acceptable salt, solvate orhydrate thereof; and (b) a second compound selected from the groupconsisting of an anesthetic, a corticosteroid, an antihistamine and ananti-inflammatory.
 22. The method of claim 21, wherein the tissue ismucosal, skin or hair tissue.
 23. The method of claim 21, wherein themethod is for protecting tissue of a human from mucositis caused byradiation and/or chemotherapeutic treatment.
 24. The method of claim 23,wherein the method is for protecting tissue of a human from xerostomiacaused by radiation and/or chemotherapeutic treatment.
 25. The method ofclaim 21, wherein the compound of formula (I) and the second compoundare administered in a single pharmaceutical composition.
 26. The methodof claim 25, wherein the single pharmaceutical composition is asolution.
 27. The method of claim 26, wherein the single pharmaceuticalcomposition is administered by injection.
 28. The method of claim 21,wherein the compound of formula (I) and the second compound areadministered in separate pharmaceutical compositions.
 29. The method ofclaim 21, wherein the compound of formula (I) and the second compoundare administered simultaneously.
 30. The method of claim 21, wherein thecompound of formula (I) and the second compound are administeredseparately.
 31. The method of claim 21, wherein the therapeuticallyeffective amount of the compound of formula (I) is 10-600 mg.
 32. Themethod of claim 31, wherein the therapeutically effective amount of thecompound of formula (I) is 100-300 mg.
 33. The method of claim 21,wherein the compound of formula (I) comprises amifostine.
 34. The methodof claim 21, wherein the second compound comprises an anesthetic. 35.The method of claim 34, wherein the anesthetic comprises pramoxine,lidocaine, procaine, mepivacaine or a combination thereof.
 36. Themethod of claim 21, wherein the second compound comprises acorticosteroid.
 37. The method of claim 36, wherein the corticosteroidcomprises hydrocortisone, hydrocortisone 21-acetate, hydrocortisone17-valerate, hydrocortisone 17-butyrate, betamethasone valerate,betamethasone diproprionate, triamcinolone acetonide, fluocinonide,clobetasol proprionate or a combination thereof.
 38. The method of claim21, wherein the second compound comprises an antihistamine agent. 39.The method of claim 38, wherein the antihistamine agent compriseshydroxyzine, diphenhydramine, propranolol, promethazine, vitamin Apalmitate or a combination thereof.
 40. The method of claim 21, whereinthe second compound comprises an anti-inflammatory agent.
 41. The methodof claim 40, wherein the anti-inflammatory agent comprises naproxen,ibuprofen, theophylline, cromolyn, albuterol, retinoic acid, 13-cisretinoic acid or a combination thereof.
 42. A method of treating damagedtissue of a human caused by radiation and/or chemotherapeutic treatment,which comprises administering: (a) a therapeutically effective amount ofat least one compound of formula (I):R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂  (I) wherein R₁ is hydrogen, C₅-C₇ aryl,C₂-C₇ acyl, or C₁-C₇ alkyl; R₂ is hydrogen, PO₃H₂ or R₃, wherein R₃ isR₁NH(CH₂)_(n)NH(CH₂)_(m)S—; and n and m are each independently aninteger from 1 to 10, or a pharmaceutically acceptable salt, solvate orhydrate thereof; and (b) a second compound selected from the groupconsisting of an anesthetic, a corticosteroid, an antihistamine and ananti-inflammatory.
 43. The method of claim 42, wherein the tissue ismucosal, skin or hair tissue.
 44. The method of claim 42, wherein themethod is for treating mucositis.
 45. The method of claim 42, whereinthe method is for treating xerostomia.
 46. The method of claim 42,wherein the compound of formula (I) and the second compound areadministered in a single pharmaceutical composition.
 47. The method ofclaim 46, wherein the single pharmaceutical composition is a solution.48. The method of claim 47, wherein the single pharmaceuticalcomposition is administered by injection.
 49. The method of claim 42,wherein the compound of formula (I) and the second compound areadministered in separate pharmaceutical compositions.
 50. The method ofclaim 42, wherein the compound of formula (I) and the second compoundare administered simultaneously.
 51. The method of claim 42, wherein thecompound of formula (I) and the second compound are administeredseparately.
 52. The method of claim 42, wherein the therapeuticallyeffective amount of the compound of formula (I) is 10-600 mg.
 53. Themethod of claim 52, wherein the therapeutically effective amount of thecompound of formula (I) is 100-300 mg.
 54. The method of claim 42,wherein the compound of formula (I) comprises amifostine.
 55. The methodof claim 42, wherein the second compound comprises an anesthetic. 56.The method of claim 55, wherein the anesthetic comprises pramoxine,lidocaine, procaine, mepivacaine or a combination thereof.
 57. Themethod of claim 42, wherein the second compound comprises acorticosteroid.
 58. The method of claim 57, wherein the corticosteroidcomprises hydrocortisone, hydrocortisone 21-acetate, hydrocortisone17-valerate, hydrocortisone 17-butyrate, betamethasone valerate,betamethasone diproprionate, triamcinolone acetonide, fluocinonide,clobetasol proprionate or a combination thereof.
 59. The method of claim42, wherein the second compound comprises an antihistamine agent. 60.The method of claim 59, wherein the antihistamine agent compriseshydroxyzine, diphenhydramine, propranolol, promethazine, vitamin Apalmitate or a combination thereof.
 61. The method of claim 42, whereinthe second compound comprises an anti-inflammatory agent.
 62. The methodof claim 61, wherein the anti-inflammatory agent comprises naproxen,ibuprofen, theophylline, cromolyn, albuterol, retinoic acid, 13-cisretinoic acid or a combination thereof.